The EBV membrane antigen (EBV-MA) gp350 contains virus neutralizing epitopes and is currently being tested as a possible EBV vaccine candidate in England. A series of monoclonal antibodies (mAbs) to identifiably different epitopes on the EBV-MA was obtained from Louis Qualtiere. Several additional mAbs were obtained from Gary Pearson. These have been used to screen antigen expressing clones for reactivity to different epitopes by ELISA to identify the nucleic acid sequences important for virus neutralization. A clone of the EBV Bam L fragment which contains the coding sequence for the gp350 in conjunction with nucleic acid sequence data obtained by computer from Genebank was used to derive appropriate constructs for gene expression in E. coli. A bacterial expression vector, pWS50, which produces fusion proteins with E. coli beta-galactosidase and a high level of protein expression, was used to generate ten overlapping clones which express different portions of unglycosylated gp350. The antigens produced by eight of these clones have been purified and used in ELISA, dot blot immunoassay and Western blot analysis with the mAbs to map the epitopes on EBV-MA. Five mAbs reacted with the recombinant E. coli antigens, and three antigenic epitopes (nucleotides 1980-2307, 3185-3527 and 35273576 in Bam HI L) identified. Protein expressed by four of the clones was used to produce antisera in rabbits. When tested for neutralization of EBV in tissue culture, the rabbit antisera as well as the mAbs which recognized the three epitopes failed to neutralize, suggesting that these epitopes are not involved in ,neutralization. The entire gp350 coding sequence has also been expressed in a Baculovirus expression system which glycosylates the protein. The properties of the Baculovirus expressed protein including its glycosylation and reactivity with mAbs are being evaluated.